Ribonucleic acid (RNA) is a single-stranded, linear polymer of nucleotides. Each nucleotide unit contains a nitrogenous base, a ribose sugar, and a phosphate group. There are several types of RNA molecules. Messenger RNA (mRNA) molecules are those whose nucleotide sequence determines the amino acid composition of proteins. In eukaryotes, the 5′-ends of most mRNAs are blocked, or “capped” with a modified guanine nucleotide. The cap contains a 5′-5′ triphosphate linkage between two nucleosides and a 7-methyl group on a guanine ring distal to the RNA polymer chain. Some other forms of RNA are also capped, e.g., small nuclear RNAs (snRNAs). RNA capping regulates intracellular molecular activities, including RNA stability and translational efficiency.
The ability to synthesize capped RNA molecules in vitro is useful because it allows one to prepare RNA molecules that will function properly in a variety of biological applications. Such applications include both research applications and commercial production of polypeptides, e.g., producing in a cell-free translation system polypeptides containing an “unnatural” amino acid at a specific site, or producing in cultured cells polypeptides that require post-translational modification for activity or stability. Because capped RNA molecules are more stable and bind more readily to the cell's translational machinery, translation of capped RNAs proceeds for a considerably longer time than is the case for non-capped RNAs, resulting in greater production of protein.
The method most frequently used to make capped RNAs in vitro is to transcribe a DNA template with either a bacterial or bacteriophage RNA polymerase in the presence of all four ribonucleoside triphosphates and a cap dinucleotide such as m7G(5′)ppp(5′)G (also called m7GpppG). The RNA polymerase initiates transcription with a nucleophilic attack by the 3′-OH of the Guo moiety of m7GpppG on the α-phosphate of the next templated nucleoside triphosphate, resulting in the intermediate m7GpppGpN. The formation of the competing GTP-initiated product pppGpN is suppressed by setting the molar ratio of m7GpppG to GTP between 5 and 10 in the transcription reaction mixture. The 5′-capped mRNAs produced with m7GpppG can take either of two forms, one containing the cap analog incorporated in the correct, forward orientation [m7G(5′)ppp(5′)GpNp . . . ], and one containing the analog in the reverse orientation [G(5′)ppp(5′)m7GpNp . . . ]. The latter are not recognized as capped mRNAs by the cell's translational machinery and decrease the translational efficiency of synthetic mRNA preparations. This problem can be averted by the use of cap analogs that have O-methyl or deoxy modifications at either the C2′ or C3′ positions of m7Guo. See J. Stepinski et al., “Synthesis and properties of mRNAs containing the novel “anti-reverse” cap analogues 7-methyl(3′-O-methyl)GpppG and 7-methyl(3′-deoxy)GpppG,” RNA, vol. 7, pp. 1486-1495 (2001); and J. Jemielity et al., “Novel ‘anti-reverse’ cap analogues with superior translational properties,” RNA, vol. 9, pp. 1108-1122 (2003). These cap analogs are incorporated into RNA transcripts exclusively in the forward orientation and are therefore called “anti-reverse cap analogs” (ARCAs). In a rabbit reticulocyte lysate (RRL) translation system, ARCA-capped mRNAs had translational efficiencies that were two-fold higher than transcripts capped with m7GpppG (Stepinski et al., 2001). In cultured mammalian cells, mRNAs capped with ARCAs are translated 2- to 2.5-fold more efficiently than those capped with m7GpppG. See E. Grudzien et al., “Differential inhibition of mRNA degradation pathways by novel cap analogs,” J. Biol. Chem., vol. 281, pp. 1857-1867 (2006).
The amount of protein produced from synthetic mRNAs introduced into cultured mammalian cells is limited by the natural degradation of mRNA. One in vivo pathway for mRNA degradation begins with the removal of the mRNA cap. This removal is catalyzed by a heterodimeric pyrophosphatase, which contains a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2). The catalytic subunit cleaves between the α and β phosphate groups of the triphosphate bridge.
E. Grudzien et al. (2006) described a cap analog, m27,3′-OGppCH2pG, in which a methylene group replaced the O atom between the α and β phosphate groups. mRNAs capped with this analog were resistant to hydrolysis by recombinant human Dcp2 in vitro. When introduced into cultured cells, mRNAs capped with the analog m27,3′-OGppCH2pG were more stable than those capped with m27,3′-OGpppG. However, the mRNA capped with m27,3′-OGppCH2pG had lower overall translational efficiency, presumably because m27,3′-OGppCH2pG has a lower binding affinity for eIF4E than that of m27,3′-OGpppG. The eukaryotic translation initiation factor eIF4E is involved in bringing the capped mRNA to the ribosome for translation.
J. Kowalska et al., “Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS,” RNA, vol. 14, pp. 1119-1131 (2008) described syntheses of three ARCAs in which one of the three non-bridging O atoms in the triphosphate chain was replaced with an S atom. Each of these phosphorothioate analogs (also called S-ARCAs) was synthesized as a mixture of diastereomers that could be separated chromatographically to make pure diastereomers. The binding affinity of the phosphorothioate cap analogs to eIF4E was equal to or, in some cases, greater than that of m7GpppG.
E. Grudzien et al., “Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells,” RNA, vol. 13, pp. 1745-1755 (2007) showed that mRNAs capped with S-ARCAs modified at the β phosphate were resistant to hydrolysis by recombinant human Dcp2 in vitro. Furthermore, mRNA capped with one β S-ARCA diastereomer had a longer half-life when introduced into mammalian cells than that of the corresponding ARCA-capped mRNA; and it also had a greater translational efficiency in cells. The first of these properties presumably resulted from the resistance of the β S-ARCA to hydrolysis by Dcp2, and the second property presumably resulted from the higher affinity of the β-S-ARCA for eIF4E.
Another use for synthetic mRNA cap analogs is to inhibit cap-dependent translation by competition with capped mRNA for binding to eIF4E. See A. Cai et al., “Quantitative assessment of mRNA cap analogues as inhibitors of in vitro translation,” Biochemistry, vol. 38, pp. 8538-8547 (1999); and E. Grudzien et al., “Novel cap analogs for in vitro synthesis of mRNAs with high translational efficiency,” RNA, vol. 10, pp. 1479-1487 (2004).
The ability of cap analogs to inhibit translation has potential therapeutic significance. Many types of cancer cells overexpress eIF4E, which can lead to increased expression of proteins that promote oncogenesis and metastasis. See A. De Benedetti et al., “eIF-4E expression and its role in malignancies and metastases,” Oncogene, vol. 23, pp. 3189-3199 (2004). Reducing eIF4E expression with siRNA, antisense oligonucleotides, or a specific eIF4E repressor can inhibit tumor growth and oncogenesis. See J. R. Graff et al., “Therapeutic suppression of translation initiation factor eIF4E expression reduces tumor growth without toxicity,” J. Clin. Investigation, vol. 117, pp. 2638-2648 (2007); and T. Herbert, “Rapid induction of apoptosis by peptides that bind initiation factor eIF4E,” Curr. Biol., vol. 10, pp. 793-796 (2000). In addition, the translational activity of eIF4E can be suppressed by saturating the cells with competing, translationally deficient cap analogs.
Some synthetic cap analogs are specific inhibitors of eIF4E activity and are therefore potentially useful as agents for treating oncogenesis and metastasis, immunosuppression in organ transplantation, and other medical conditions. However, these potential uses for cap analogs have never previously been demonstrated in vivo, in part due to the instability of cap analogs in intracellular conditions.
J. Kowalska et al. (2008) demonstrated that γ-S-ARCAs are strong inhibitors of translation in a cell-free system, presumably due to their high binding affinity for eIF4E. The γ-modified analogs are resistant to hydrolysis by the human DcpS enzyme, which is a scavenger pyrophosphatase responsible for degradation of this type of compound.
Other modifications can help protect capped mRNA against enzymatic degradation. One example is a boranophosphate modification, in which one of the non-bridging O atoms is replaced with a borane group (BH3−) (sometimes called the BH3-analogs). Another example is a phosphoroselenoate modification, in which one of the non-bridging O atoms is replaced with a selenium atom (sometimes called the Se-analogs). The phosphorothioate, boranophosphate, and phosphoroselenoate groups all replace non-bridging oxygen atoms and share some chemical and biochemical properties. However, there are also differences among these groups. For example, the P—X bond lengths differ (where X denotes S, Se, or BH3), the van der Waals radii of the X groups differ, and the affinity of the X groups for various divalent and other metal cations differ. These differing chemical properties alter the biological activities of cap analogs with these groups, including their interactions with cap-binding proteins and their susceptibility to enzymatic degradation.
Boranophosphate mononucleotides and boranophosphate polyphosphate dinucleotides were reviewed by P. Li et al., “Nucleoside and oligonucleoside boranophosphates: chemistry and properties,” Chem. Rev., vol. 107, pp. 4746-4796 (2007).
Boranophosphate polyphosphate dinucleoside analogs are described in published patent application US2006/0287271, as are their use against diseases modulated by P2Y receptors, e.g., type 2 diabetes and cancer.
Boranophosphate nucleotide analogs have similarities with phosphorothioate analogs due to similar bond angles, pKa values, and P-diastereoisomerism. However, in some cases they are as much as 10-fold more resistant to enzymatic hydrolysis than their phosphorothioate counterparts. They are also more lipophilic than phosphorothioates, which may help them to penetrate cell membranes and reach intracellular translational machinery. Boranophosphate analogs can also be used in boron neutron capture therapy (BNCT). See B. R. Shaw et al., “Reading, Writing and Modulating Genetic Information with Boranophosphate Mimics of Nucleotides, DNA and RNA,” Ann. N.Y. Acad. Sci., vol. 1201, pp 23-29 (2003); and J. Summers et al., “Boranophosphates as Mimics of Natural Phosphodiesters in DNA”, Current Medicinal Chemistry, vol. 8, pp 1147-1155 (2001).
Phosphoroselenoate analogs of nucleoside di- and triphosphates modified in the α position were described in K. Misiura et al., “Synthesis of nucleoside α-thiotriphosphates via an oxathiaphospholane approach,” Org. Lett., vol. 7, pp 2217-2220 (2005); P. Li et al., “Synthesis of α-P-modified nucleoside diphosphates with ethylenediamine,” J. Am. Chem. Soc., vol. 127, pp. 16782-16783 (2005); N. Carrasco et al., “Enzymatic synthesis of phosphoroselenoate DNA using thymidine 5′-(α-P-seleno)triphosphate and DNA polymerase for x-ray crystallography via MAD,” J. Am. Chem. Soc., vol. 126, pp. 448-449 (2004); and N. Carrasco et al., “Efficient enzymatic synthesis of phosphoroselenoate RNA by using adenosine 5′-(α-P-seleno)triphosphate,” Angew. Chem. Int. Ed., vol. 45, pp. 94-97 (2006). However, to the knowledge of the inventors, there have been no prior reports of nucleoside polyphosphate analogs modified in any position other than the α position, nor of dinucleoside polyphosphates modified at any position.
Phosphoroselenoate nucleotide analogs are similar to phosphorothioates and boranophosphates due to their similar bond angles, pKa values, P-diastereoisomerism, and resistance to enzymatic degradation. Phosphoroselenoates can be very useful in nucleic acid crystallography because Se can be used in the multi-wavelength anomalous dispersion (MAD) technique. See J. Wilds et al., “Selenium-assisted nucleic acid crystallography: use of phosphoroselenoates for MAD phasing of a DNA structure,” J. Am. Chem. Soc., vol. 124, pp 14910-14916 (2002); N. Carrasco et al. (2004); N. Carrasco et al. (2006); and P. S. Pallan et al., “Selenium modification of nucleic acids: preparation of phosphoroselenoate derivatives for crystallographic phasing of nucleic acid structures,” Nat. Protoc., vol. 2, pp. 640-646 (2007).
See also our work on anti-reverse cap mRNA analogs described in U.S. Pat. No. 7,074,596; and published international patent application WO 2008/157688.